1. Field of the Invention
The present invention relates to a method and apparatus for distinguishing and identifying subpopulations of cells in a sample without interference from other cells or particles in the same, and more particularly, related to a method and apparatus for distinguishing and quantifying blood cells or subpopulations of blood cells in a sample without interference from other blood cells, particularly red blood cells.
2. Description of the Prior Art
The leucocyte (white blood cell) population in blood is defined by a number of subclasses which play distinct roles in the immune response. For example, the relative number of cells in various lymphocyte subclasses is likely to change in disease states. Hence, identification of cells of the various subclasses provides an analytic tool that has been found to be of great value in the diagnosis of various diseases and the monitoring of treatment for the disease.
It is known that at least several particular subclasses of functionally distinct lymphocytes can be identified by antibodies, particularly monoclonal antibodies, to antigenic determinants on the cell surface. In particular, there has been considerable academic and clinical interest in the identification of the various subclasses of T-lymphocytes. T-lymphocytes are characterized by identifiable antigenic determinants on their surface which distinguish the cells of that subclass from other blood cells and from the cells of other lymphocyte subclasses. Hence interest is high in developing rapid methods for the analysis of white blood cells, particularly the analysis of lymphocytes and the T-cell subclass of lymphocytes.
T cells are divided into at least several subtypes, termed "helper", "suppressor", and "cytotoxic" T cells, which have the function of (respectively) promoting a reaction, suppressing a reaction, or killing (lysing) foreign cells. The ability to identify subtypes of T cells is important for diagnosis or treatment of various immunoregulatory disorders or conditions.
The preparation of a blood sample for analysis of the leucocyte (white blood cell) populations has usually been based on the apparent need to separate the white blood cells from the erythrocyte (red blood cell) population. The red blood cells outnumber the white blood cells by a factor of about 1000 to 1. The overwhelming prominence of red blood cells has led investigators to conclude that the red blood cells must be removed from the sample prior to an effective analysis of the white blood cells in the sample by flow cytometry techniques.
In general, two different methods have been developed for providing a white blood cell sample free from interference from red blood cells. IN one method, the white blood cells are isolated and separated from the red blood cells by centrifugation in the presence of a partitioning agent, such as Ficoll-Paque (.TM.), which is marketed by Pharmacia Fine Chemicals AB, Upsala, Sweden, or a thixotropic gel-like substance. In another method, described in U.S. Pat. No. 4,284,412 to Hansen et al., the red blood cells are lysed to break out the red blood cells into fragments which do not interfere with flow cytometry analysis.
The initial step of separating white blood cells or a subclass thereof from red blood cells is long and arduous; in fact much longer than the relatively simple steps of staining or analyzing the white blood cells or subclass thereof. For clinical purposes, wherein there exists a considerable premium on rapid, repetitive analyses, the necessity of isolating the white blood cells from red blood cells and undesired subclasses of white blood cells is a serious impediment. Furthermore, even for those research applications wherein time is at less of a premium, an initial separation step involves the risk of loss of some of the desired white blood cells during removal of the red blood cells and undesired white blood cells, which introduce uncertainty and inaccuracy to the subsequent analysis. Correspondingly, failure completely to eliminate undesired blood cells from the sample to be analyzed introduces considerable risk of error, and the presence of such undesired cells may well cause errors in the subsequent analysis. Methods involving lysis of the red blood cells are also not wholly satisfactory in that it is difficult to obtain lysis of all the red blood cells without incurring some lysis of the white blood cells.
Presently known and available flow-through cytometers useful for detecting particles, cells and the like, commonly include two channels for the detection of one or more, usually two, subpopulations of cells in a mixture. For example, devices are known which include two fluorescence channels which can detect cells specifically labeled with two immunofluorescent agents associated with the respective fluorescence channels. In these known devices, a complete fluorescence channel including the electrical circuitry and fluorescence detector is used for each category of fluorochrome-treated cells to be detected in the mixture of cells in the sample being analyzed. Therefore, to detect multiple subpopulations of cells in a sample using flow-through cytometry, an equivalent number of fluorescence channels is used. A separate light source, such as a laser, may be used to excite each different type of fluorochrome or immunofluorescent stain which has been tagged. Apparatus utilizing two lasers for analyzing immunofluorescent stains are described in U.S. Pat. Nos. 3,826,364 and 4,284,412.
Analysis and quantifying of two different immunofluorescent stains in a single sample can also be performed with two or more fluorochromes which are activated by a single light source but which have different emission characteristics.
In the field of hematology in general, and in the specific field of immuno-hematology, it is desirable to determine the count of a variety of white blood cells which circulate in the peripheral blood. Subclassification of cells is performed, and the count of the subclasses is of great interest in the evaluation of immune related disease, such as the recently recognized acquired immune deficiency syndrome (AIDS). In particular, the subclasses of the lymphocyte, a mononuclear type of white blood cells in blood, has become of great clinical significance.
There are many instances when it is desirable to be able to directly detect distinct multiple subpopulations of white blood cells of interest from a sample. For instance, in performing certain tests on blood, it may be desirable to detect or quantify the total population of lymphocyte cells in the blood sample and to determine the proporation of T-cells and B-cells as a percentage of the lymphocyte population. Similarly, the detection and quantification of other different types of lymphocytes, such as the helper cell/suppressor cell subset of T-cells and the suppressor cell/natural killer cell subset of T-cells, may be desired. With this in mind, the present invention is directed to satisfying the desired need for the direct determination of subpopulations of white blood cells of interest from a sample mixture without interference from red blood cells.
It is, accordingly, a primary object of the present invention to provide a method for identifying and enumerating a set of blood cells, particularly white blood cells, and specific subclasses of the set of blood cells, while avoiding the necessity for prior separation or lysis of interfering blood cells, particularly red blood cells. It is another object to provide a method for analysis of white blood cells and subclasses thereof which substantially prevents faulty analysis due to the presence of other cells, such as red blood cells, or loss of data through loss of cells from the sample. It is a further object to provide such methods wherein the speed and relative simplicity involved renders blood cell subclass identification and enumeration a viable clinical tool.